Allogeneic hematopoietic cell transplantation (HCT) is a form of cancer immunotherapy that relies on donor T cells to facilitate engraftment and induce the beneficial the graft-versus-leukemia effect. However, alloreactive donor T cells can also cause fatal graft-versus-host disease (GVHD). Genetic modification of donor T cells with a "suicide" gene, herpes simplex virus thymidine kinase (HSV-tk), has the potential to significantly improve the safety of allogeneic HCT by killing the alloreactive, GVHD-causing T cells with ganciclovir. To date, clinical studies of HSV-tk modified donor T cells infused after allogeneic HCT have shown very limited in vivo function. We propose a novel strategy to favor the in vivo immune function of genetically modified T cells in a large animal model of allogeneic HCT. The hypothesis is that lentiviral transduction of donor T cells with an immunosuppression drug-resistance gene conferring a selective proliferative advantage over host immune cells can significantly improve the in vivo function of the gene-modified T cells. We will evaluate a bicistronic lentiviral vector expressing HSV-tk and a dominant mutant inosine monophosphate dehydrogenase II (IMPDH*) gene, which renders transduced T cells sensitive to ganciclovir and resistant to the immunosuppressive drug mycophenolate mofetil (MMF). Using the dog model of MHC-mismatched HCT, we will test the in vivo function of MMF-resistant donor T cells to facilitate engraftment of T-cell-depleted (TCD) donor marrow after total body irradiation (TBI). Treatment of recipients with MMF after infusion of IMPDH* transduced donor T-cells would inhibit immune responses to the gene modified T cells and give a proliferative advantage to the transduced T cells. This approach allows the graft-versus-host function of the MMF-resistant donor T cells while suppressing the host-versus-graft response. Aim 1 will study the in vivo effectiveness of the IMPDH* transduced donor T cells to facilitate engraftment after myeloablative TBI. Dogs with GVHD will be treated with ganciclovir. Aim 2 will decrease the TBI dose needed to engraft TCD marrow by treating with a combination postgrafting cyclosporine and MMF and infusion of MMF- and cyclosporine-resistant T cells. After engraftment, ganciclovir will be given to control GVHD. The theme of this project is to apply a gene therapy strategy of immunosuppressive drug resistance to manipulate the immune function in favor of donor immune cells after HCT. Results from these studies have the strong potential to be directly translated to future gene therapy clinical trials.